Environ. Sci. Technol.

2000, 34, 1081-1087

Environment Act, increases in landfill tax. and a commitment

An Integrated Method Incorporating by the U.K. government to build 60% of new houses onSulfur-Oxidizing Bacteria and previously developed sites (2) have increased the pressure to develop effective remediation technologies. Over 60% ofElectrokinetics To Enhance Removal sites are co-contaminated with metals and organics. The metals are frequently found as complex mixtures. and manyof Copper from Contaminated Soil former industrial sites contain highly toxic metals such as cadmium, nickel. and arsenic. Therefore, in the U.K., as inGIACOMO MAINI,§ AJAY K. SHARMAN,§ other countries, the potential for developing these sitesGARRY SUNDERLAND,‡ together with the possibility that metals may leach intoCHRISTOPHER. J. KNOWLES,† AND groundwater or enter the food chain through plant materialS I M O N A . J A C K M A N * ,† means that remediation of metal-contaminated land is aIBS-Viridian Ltd., 114-116 John Wilson Business Park, priority. There are currently few comprehensive remediationThanet Way, Whitstable, Kent CT5 3QT, U.K., EA Technology technologies available for such sites.Ltd., Capenhurst, Chester CH1 6ES, U.K., Oxford Centre for Within the mining industries, technologies have beenEnvironmental Biotechnology, Department of Engineering developed for the recovery of valuable metals from ores andScience, University of Oxford, Parks Road, Oxford OX1 3PJ, rock/soil materials. Indeed the use of bacteria in bioleachingand NERC Institute of Virology & Environmental has become a prominent method of recovery (3). NaturallyMicrobiology, Mansfield Road, Oxford OX1 3SR, U.K. occurring iron- and sulfur-oxidizing bacteria from mining wastes have been isolated and strains selected for their ability to solubilize metals from different substrates (4). In this respect, the bacteria involved in bioleaching processes areThe combination of bioleaching and electrokinetics for able to convert metal sulfides to their respective sulfates,the remediation of metal contaminated land has been thereby transforming them from insoluble to soluble salts.investigated. In bioleaching, bacteria convert reduced sulfur The metals can be recovered by washing and furthercompounds to sulfuric acid, acidifying soil and mobilizing processing. This established technology is now being ex-metal ions. In electrokinetics, DC current acidifies soil, and tended to investigate the leaching of contaminating metalsmobilized metals are transported to the cathode by from soils as a bioremediation technique (5). The application of mixed cultures of bacteria has proved successful inelectromigration. When bioleaching was applied to silt mobilizing metals within different soils. Difficulties, however,soil artificially contaminated with seven metals and amended may be presented by inhibition of microbial activity due towith sulfur, bacterial activity was partially inhibited and the mobilized metals, with the potential for the entirelimited acidification occurred. Electrokinetic treatment of bioleaching process to come to a halt (6). The presence ofsilt soil contaminated solely with 1000 mg/kg copper nitrate anionic species is also known to inhibit sulfur oxidation asshowed 89% removal of copper from the soil within 15 pH values are reduced (7). The potential for metals to leachdays. To combine bioleaching and electrokinetics sequentially, from soils means that to avoid groundwater contaminationpreliminary partial acidification was performed by the process has either to be conducted ex situ, or, if in situ,amending copper-contaminated soil with sulfur (to 5% with a metal removal system.w/w) and incubating at constant moisture (30% w/w) and Electrokinetics is an emerging engineering technique fortemperature (20 °C) for 90 days. Indigenous sulfur- the remediation of contaminated land (8-10). The applica- tion of a direct current to soil, by insertion of electrodes,oxidizing bacteria partially acidified the soil from pH 8.1 to leads to the generation of hydrogen ions at the anode and5.4. This soil was then treated by electrokinetics yielding hydroxyl ions at the cathode. These migrate into the soil86% copper removal in 16 days. In the combined process, and the hydrogen ions can displace adsorbed metal ionselectrokinetics stimulated sulfur oxidation, by removing into the pore fluid of the soil. By manipulation of theinhibitory factors, yielding a 5.1-fold increase in soil sulfate conditions surrounding the cathode, an acidic pH can beconcentration. Preacidification by sulfur-oxidizing bacteria maintained throughout the soil. Metal ions, once solubilized,increased the cost-effectiveness of the electrokinetic can be transported by electromigration through the soil andtreatment by reducing the power requirement by 66%. recovered at the cathode. The process has been effective in both model and real systems and has been applied to sites in the United States (11) and in Europe (12). The combination of bioleaching and electrokinetics hasIntroduction the potential to overcome several of the limitations of theIn the U.K. alone, 200 hundred years of industrialization has individual techniques with the possibility that some of theresulted in an estimated 200 000 contaminated sites (1). A combined attributes may prove to be synergistic. In particular,significant number of these contain cocktails of toxic electrokinetics mobilizes metal ions provided their speciationchemicals that provide a hazard to both human health and is appropriate. Metals as hydroxides or oxides may bethe environment. The Confederation of British Industry has solubilized by the electrokinetic acidification, but thoserecently estimated the cost of remediating this contaminated present as insoluble sulfides, a common speciation in formerland at £ 20bn (1). The implementation of the U.K. 1995 gasworks sites and mining wastes, will not be extracted by this method. However, the bacteria involved in bioleaching * Corresponding author phone: +44 1865 281630; fax: +44 1865 processes can convert metal sulfides to sulfates, thereby281696; e-mail: saja@wpo.nerc.au.uk. § IBS-Viridian Ltd. enabling their solubilization and subsequent transport by ‡ EA Technology Ltd. electromigration. In addition, the directional transport of † University of Oxford and NERC Institute of Virology & Envi- metal ions by electrokinetics is a useful complement toronmental Microbiology. bioleaching as solubilized metals can be removed at the10.1021/es990551t CCC: $19.00  2000 American Chemical Society VOL. 34, NO. 6, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 1081Published on Web 02/17/2000 preliminary bioleaching experiments with static soil and soilTABLE 1. Characteristics of Standardized Silt Soil slurries, it was decided to focus upon a single contaminating parameter value metal for electrokinetic studies. This would enable a clearer elucidation of the complementary effects of bioleaching and pH 8.1 electrokinetic metal removal. Copper was chosen as the soil density (kg/dm3) 1.350 contaminant and applied as 1000 mg/kg copper nitrate. extractable phosphorus (mg/L) 24 particle size, diameter (mm) <2 Static Soil Experiment. Contaminated and uncontami- cationic exchange capacity (me/100 g) 21.7 nated soils were amended with sulfur (mesh size 50) at two soil buffering capacity (g/kg) (calcd as the 0.52 different concentrations (0.5 and 5% w/w) by slow addition amount of [H+] from sulfuric acid of sulfur with constant mixing. Water was added to 30% (w/ required to acidify the soil to pH 2) w). Soils were set aside in trays with a soil depth of 10 cm permeability, k (m/s) 1.81 × 10-11 at constant temperature (20 °C) and moisture (maintained copper concn (mg/kg) 35.5 by regular dry weight measurement and addition of water) calcium carbonate (%) 2 for up to 42 days. After 21 and 42 days, samples were taken total sulfur (%) 0.08 and analyzed for pH (by suspending 1 g of soil in 10 mL of total nitrogen (%) 0.25 organic matter (%) 3.40 water) and the concentration of sulfate. Sulfate analysis was organic carbon (%) 1.97 by addition of 10 mL of 0.1 M HCl to 1 g of soil and mixing. C:N ratio 7.9:1 Following centrifugation, barium chloride was added to samples, precipitating barium sulfate, which was analyzed by spectrophotometry.cathode for straightforward downstream processing. A further Soil Slurry Experiment. Soil slurries were prepared fromadvantage is the potential for preliminary bioleaching prior contaminated and uncontaminated silt soil amended withto electrokinetics to reduce the overall time scale and cost sulfur and added to deionized water at 30% (w/v) to give aof electrokinetic remediation. By amendment of the soil with total volume of 300 mL in 500 mL conical flasks. Slurriesinexpensive sulfur (below $ 250/tonne) and simply allowing were incubated with shaking (160 rpm) at 30 °C, and samplesthe soil bacteria to metabolize, predictable sulfate generation were taken for analysis of pH and sulfate concentration.and acidification can be achieved. Little intervention, in terms Electrokinetic Studies. The apparatus for performingof man hours, is required in comparison to acid washing of electrokinetics is outlined in Figure 1. A soil cell system wassoil with relatively more expensive and hazardous chemicals constructed with six individual compartments each being(e.g. sulfuric acid). This preliminary bioleaching stage forms 6.5 cm long between anode and cathode, 3 cm wide and 4an important part of the current study. cm deep, and containing approximately 150 g of moist soil. Electrokinetics together with bioremediation has been This arrangement allowed samples to be taken at a range ofused to effect the movement and degradation of phenol by time points. Within each compartment, soil could be slicedan industrial/government consortium in the United States into five centimeter-wide sections to enable measurements(13). The same consortium has developed this technology of pH, moisture content, and metal ions. pH was determinedup to a full-scale field test to move trichloroethylene (TCE) by suspending 1 g of soil in 10 mL of water and using ainto treatment zones in which it is degraded by reaction with standard pH meter. Moisture content was determined byzerovalent iron (14). The introduction of nutrients into soil drying soil at 110 °C for 24 h and comparing wet and dryby electrokinetics (15, 16) and movement of other organics weights. Metal ions in soil were analyzed by Electronhave also been investigated (17), but there has been little use Diffraction X-ray Fluorescence (ED-XRF; CPL Laboratories,of combined techniques for remediation of metal contami- Derbyshire, U.K.). Anodes were of carbon felt material (8.5nation. The behavior of sulfur-oxidizing bacteria at acidic cm wide, 7 cm deep, and 1.5 cm thick), and stainless steelpH values has been well studied (18), and the acidophilic mesh (14 cm long and 5 cm wide folded in the center) wasbacterium Thiobacillus ferrooxidans has been used for used for cathodes. Each cathode was located in a compart-combined electro/bioleaching processes for metal recovery ment (8.5 cm wide, 7 cm deep containing approximately 80(19, 20). The paucity of data concerning microbial activities mL of liquid) bordered by a semipermeable membrane toin soil in electric fields led us, therefore, to investigate the allow ions and water to pass from soil to cathode. This enabledbehavior of sulfur-oxidizing bacteria and heterotrophs in the a catholyte solution to be contained within the cathodepresence of a DC current. We established that, in the presence compartment and recirculated through an ion exchangeof soil, both Thiobacillus thiooxidans-like organisms and column for recovery of metals. The total volume of theheterotrophs were protected from the otherwise deleterious catholyte system was approximately 500 mL with a recir-effects of the electric current and indeed stimulation of culation rate of approximately 5 mL/min. A pH control unitmetabolic activity was observed (21). was incorporated to maintain an acidic pH (pH 4.5) in this The purpose of the current study is to combine soil compartment through addition of acetic acid. An aqueousacidification by indigenous sulfur-oxidizing bacteria with feed was attached to the anode, and electrical connectionselectrokinetic remediation for removal of copper ions from were attached to a direct current power pack.contaminated soil. A first series of experiments used silt soil contaminated with 1000 mg/kg Cu(NO3)2 and adjusted to 30% (w/w)Experimental Section moisture. The experiment shown here is a representative ofAll studies were conducted using a standardized silt soil a series of three studies which were conducted under identicalobtained from Wye College, University of London (see Table conditions and yielded the same results. A current density1 for characteristics). The soil was contaminated by spraying of 3.72 A/m2 electrode surface area was applied at a constanta solution of metals onto the soil and mixing to effect a current with 8 M acetic acid added to the catholyte tohomogeneous contamination. For preliminary studies, a maintain an acidic pH, and 0.05 M H2SO4 was added to thecocktail of six metals was used, added as nitrates, at anode at 60 mL/day.concentrations of 10 mg/kg Cd, 500 mg/kg Cr, 500 mg/kg In a second set of experiments, the same soil was amendedCu, 200 mg/kg Ni, 2000 mg/kg Pb, and 2000 mg/kg Zn, plus with 5% (w/w) sulfur and 30% (w/w) moisture and allowed200 mg/kg molybdate. These concentrations were chosen to incubate for 90 days at 20 °C prior to electrokinetics. Twosuch that the soil was characterized as contaminated electrokinetic experiments were conducted using this soil:according to U.K. government guidelines (22). Following these one with 3.72 A/m2 current density and the other with the

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FIGURE 1. Electrokinetic apparatus. The power pack was able to deliver up to 2 A and 60 V. Acid reservoir contained 8 M acetic acid.The pH control unit was set to add acetic acid when the pH in the catholyte reservoir, as measured by a pH probe, increased above pH4.5. Recirculating pumps were used to maintain a flow of catholyte through the ion exchange column and the mixing reservoir at approximately5 mL/min. Anolyte feed contained either 0.05 M sulfuric acid (first run) or water (second run) and was supplied at approximately 60 mL/day.The box containing the anode and cathode was also filled with soil, and the movements of water and metal ions are shown.

The pH profile shows that in uncontaminated soil

TABLE 2. pH and Sulfate Contents of Metal-Contaminated and amended with 5% (w/w) sulfur acidification to approximatelyUncontaminated Soils Amended with 0.5 and 5% (w/w) Sulfur pH 1 could be achieved in 55 days. However, when this soil(S) and Incubated at 20 ˚C at 30 % (w/w) Moisturea was contaminated with metals, the pH was only reduced to 21 days 42 days pH 2.5 over this time scale. Amendment of uncontaminated sulfate sulfate and contaminated soils with 0.5% (w/w) sulfur (results not sample pH mg/kg soil pH mg/kg soil shown) led to acidification to pH 4 in both cases. Sulfate generation above 2000 mg/kg was only seen with uncon-0.5% S, uncontaminated 4.87 4910 4.31 8590 taminated soil amended with 5% (w/w) sulfur. In all other0.5% S, contaminated 6.46 820 6.39 1200 flasks in which sulfur was added, sulfate generation reached5% S, uncontaminated 5.01 5120 3.38 13770 1000-1500 mg/kg. This lower level of sulfate was sufficient5% S, contaminated 6.28 1640 7.05 1320 to reduce the pH of the soil to between 2.5 and 4.5, but it wascontrol 7.78 40 7.74 0 not sufficient for further acidification. Separate analyses of a The control soil was not amended with sulfur but was maintained microbial populations by plating techniques indicated thatat constant (30% w/w) moisture for the duration of the experiment. ThepH of the soil at the start of the experiment was 8.1. during the transition from pH 8 to pH 4, the predominant species of sulfur-oxidizing bacteria present were neutrophilic in terms of their growth pH (S. A. Jackman, unpublishedcurrent density increased to 7.44 A/m2 after 7 days. In both results), whereas below pH 4, acidophilic bacteria, similar tocases, 8 M acetic acid was added to the catholyte, and Thiobacillus thiooxidans, predominated (21). It has beendeionized water was added to the anode at 60 mL/day. demonstrated that at lower pH values metal ions such as Metal was recovered using a column packed with ion- copper and zinc become soluble as ionic species in soil (23).exchange resin (Amberlite IRC-718). The column was re- At this point, therefore, their toxicity to soil bacteria mayplaced when copper adsorption became visible. Copper was increase. Nitrates and other anions are also more toxic toeluted from the ion-exchange resin using a 15% (v/v) HCl sulfur oxidizing bacteria at low pH values, due to theirsolution and analyzed by Atomic Absorption Spectroscopy protonation and potential movement into cells, destroying(AAS). The total run times were 15 and 16 days for the first the membrane potential, ∆ψ (7). These toxic effects couldand second experiments, respectively. Soil metal concentra- cause the reduction in sulfur metabolism by indigenoustions were determined by ED-XRF. sulfur-oxidizing bacteria under these conditions. As a link to the electrokinetic experiments, which wereResults performed upon soil containing a single metal contaminant, this copper-contaminated soil (1000 mg/kg copper nitrate)Static Soil Experiment. Contaminated and uncontaminated was tested in a soil slurry (5% w/v) containing 10% (w/w)silt soils were amended with sulfur at 30% (w/w) moisture sulfur. Similar profiles for pH and sulfate generation wereand incubated (Table 2). found as with the soil contaminated with a cocktail of seven In the case of soil that had not been contaminated by metals.addition of a cocktail of metals, sulfate was produced, and Electrokinetic Experiments. Standard silt soil contami-the pH was reduced, whereas with metal-contaminated soil, nated to 1000 mg/kg with Cu(NO3)2 was subjected tosulfate production was lower and the pH of the soil was not electrokinetics. No sulfur was added to the soil, and thereforeas much reduced. With 5% sulfur amendment, there was a no bacterial sulfur oxidation would be expected. The data in10-fold difference in the level of sulfate production between terms of metal recovery at the cathode and overall pH of thecontaminated and uncontaminated samples, which was soil are displayed in Figure 3.accompanied by a pH difference of approximately 3.7 units. Over 90% of copper ions were removed at the cathode Soil Slurry Experiment. Silt soil was amended with sulfur within 15 days of initiation of the electrokinetic treatment.to 5% (w/w) and made into a slurry at 30% (w/v). The results Similarly, there was removal of 70-90% of the calcium andin terms of the change in pH and sulfate production for metal- manganese. Analyses of magnesium and iron were alsocontaminated and uncontaminated soils are shown in Figure performed, but their movement was negligible under the2. electric field. The soil temperature was between 25 and 35

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FIGURE 2. Acidification and sulfate production in silt soil slurries amended with sulfur. Uncontaminated silt soil was compared to soilcontaminated with a cocktail of seven metals. Data points for uncontaminated soil are shown by solid symbols and those for contaminatedsoil by open symbols. Each flask (500 mL) contained 300 mL of 30% (w/v) soil slurry not amended with sulfur (diamonds) or amended with5% w/w sulfur (squares). Solid lines are for pH values and dotted lines for sulfate concentration.

FIGURE 3. Metal recovery from the catholyte and the change in pH of the soil during electrokinetics. Recoveries are expressed as apercentage of the starting metal concentrations in the soil. Copper ions are shown as triangles, calcium as squares, and manganese ascircles. pH is shown in diamonds with dotted lines.

°C. The operating voltage remained at an average of 12.2 V significantly below the buffering capacity for the soil,for the first 9 days of the experiment, rising to an average of indicating that the acidic pH was as a result of the electro-38.0 V for the remaining 6 days. The operating current was kinetics. Measurement of chloride and acetate concentrations100 mA, and the average voltage over the whole experiment demonstrated migration of these ions toward the anode.was 23.0 V. Soil moisture content was maintained at between Chloride concentration was reduced from 818 mg/kg to less20 and 25% (w/w) over the course of the experiment. Water than 30 mg/kg across the soil within 24 h. Acetate concen-balance determinations showed that 91% of water added to tration in the soil increased as the ion migrated into it fromthe apparatus, in terms of soil moisture and liquid added to the catholyte. After 7 days, concentrations of approximatelythe electrodes, was recovered at the end of the experiment. 10 000 mg/kg were determined across the whole area between The pH and copper profiles across the soil, which was the anode and cathode.divided into five centimeter-wide sections from anode to A second experiment was conducted, also with silt soilcathode, were determined and are shown in Figure 4. contaminated with copper nitrate to 1000 mg/kg and with The mass balance for copper was 100.1% as recovered the moisture adjusted to 30% (w/w), but, in this case, sulfurwithin soil samples (assayed by ED-XRF) plus from the was added to 5% (w/w). In this experiment, the soil wascatholyte by ion exchange (assayed by AAS). Addition of preincubated for 90 days at 20 °C prior to the application ofsulfuric acid (approximately 86 mg/kg soil) at the anode was DC current, during which time the pH of the soil reduced

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FIGURE 4. Copper concentration and pH profile across soil during electrokinetics of silt soil contaminated with 1000 mg/kg copper nitrate.Individual time points are shown for time 0 (squares), 2 days (diamonds), and 15 days (circles). Solid lines are for copper concentrationand dotted lines for pH.

whereas iron and magnesium showed negligible movement.

Sulfate appeared to concentrate within the center of the electrokinetic apparatus, but, as the electrokinetics pro- gressed, it moved toward and into the anode compartment. Initially, the soil contained 3101 mg/kg sulfate. After 10 days, the level increased to an average of 15 939 mg/kg, an increase of 5.1-fold. Within this same 10-day period, the average copper concentration in the soil was reduced from 1000 mg/ kg to 186 mg/kg. After this time, the sulfate content of the soil reduced to an average of 6417 mg/kg after 16 days as the sulfate migrated into the anode compartment. Movements and concentrations of chloride and acetate were similar to those in the previous experiment. An identical experiment in which the relative current density was increased to 7.44 A/m2 after 7 days led to greaterFIGURE 5. Sulfate concentration across soil during electrokinetics. movement of sulfate ions toward the anode such that theThe soil was sectioned into five equal portions, the data for which soil was cleared to less than 1000 mg/kg sulfate between theare shown at 1-cm distances from the anode. Results (( SEM) are electrodes.displayed for time 0 (squares), 3 days (diamonds), 10 days (circles),and 16 days (triangles). The power consumption for each of the electrokinetic runs could be calculated from the applied voltage and currentfrom 8.1 to 5.4 due to the activity of the sulfur-oxidizing and the run time. For the first experiment the total electrodebacteria. Average sulfate production was 3101 mg/kg soil. surface area was 0.0162 m2. With an operating current of 0.1After the incubation period, the soil was packed into an A, this yields a current density of 6.2 A/m2. The average appliedelectrokinetic reactor, and a DC current was applied under voltage was 23 V, over a distance of 6.5 cm between thethe same conditions as for the previous experiment with the electrodes. Assuming a linear increase in voltage at increasingexception that water was added to the anode as opposed to distance between the electrodes, this gives a voltage of 354sulfuric acid. Copper removed from the soil was collected by V/m. Power consumption was therefore 786 kWh/m3. At aion exchange, and soil samples were taken during the course power cost of $0.05/kWh and total run time of 15 days,of the experiment. Analyses of the soil pH and copper assuming 1 m3 of soil is approximately 1.5 tonne, the totalconcentration revealed very similar profiles to those displayed cost of metal removal was therefore $26.2 per tonne. Whenin Figure 4, with pH reduced to below 3.0 in 9 days. The the soil was preincubated with sulfur-oxidizing bacteria,average copper concentration across the soil was 114 mg/kg power consumption was 281 kWh/m3, yielding a cost of $9.3after 16 days. Soil moisture content was maintained at per tonne, a cost reduction of 66%. The cost of sulfur, atbetween 24 and 31% (w/w), and the soil temperature was below $250/tonne, and a relatively low input in terms ofbetween 20 and 27 °C over the course of the experiment. The man hours for monitoring and sampling do not greatly affectapplied voltage remained at an average of 6.3 V for the course this cost reduction.of the experiment, apart from a peak of 28.2 V at day 8. Overall,the average voltage was 7.7 V, with an operating current of Discussion100 mA. Figure 5 shows the sulfate profile across the soil The potential of combining bioleaching by sulfur-oxidizingduring electrokinetics. bacteria with electrokinetics for metal mobilization and The removal of copper from the soil at the cathode was recovery has been investigated. Initial experiments with sulfur80% after 10 days and 86% after 16 days treatment. The final amendment of contaminated soil demonstrated that acidi-mass balance for copper was 108%. Calcium and manganese fication was partially inhibited in comparison with uncon-were also readily mobilized and removed from the soil, taminated soil in both static soil experiments and in soil

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slurries. Static experiments showed continuing production acidification of the soil below pH 4.5. This appeared toof sulfate and reduction in pH in uncontaminated soil up to overcome the resistance which the sulfur-oxidizing bacteria42 days. In comparison, after 21 days, there was no further encounter in attempting to reduce the pH of the soil to belowincrease in sulfur oxidation in contaminated soil amended pH 3. The rise in sulfate production accompanied removalwith 5% sulfur. In soil slurries, enhanced sulfur oxidation of copper ions from the soil by electrokinetics. Experimentaland acidification might be expected due to increased aeration studies have also demonstrated that nitrate is rapidly removedand dilution of the soil with water. While a reduction in pH (by electromigration toward the anode) from silt soil byto below pH 3 was observed, in comparison to no significant electrokinetics (S. A. Jackman, unpublished results), althoughpH change in static experiments, sulfate production remained it is also possible to speculate that microaerophilic sulfurbelow 2000 mg/kg indicating that inhibition of bacterial oxidizers were able to reduce nitrate in the soil, therebyactivity was still occurring. Inhibition of bacterial metabolism relieving its inhibitory effects. It is possible that this removalby metal ions has been studied for a wide range of bacteria of inhibitory metals and anions was sufficient for the activity(23), and with acidophilic sulfur-oxidizing bacteria, copper of the sulfur-oxidizers to be stimulated. However, the rate ofand other ions have been shown to be inhibitory (24), sulfate production in the soil reached an average of 894 mg/although the concentration of copper in this experiment may kg/day between days 3 and 7 and 1311 mg/kg/day betweennot have been sufficient to produce the level of inhibition days 7 and 10. In comparison, sulfate production in un-observed. Anions, especially nitrate, have also been shown contaminated soil in the static soil experiment was only 328to be inhibitory at acidic pH values (7). Nitrate concentrations mg/kg/day. Clearly, the rate of sulfate production waswere significant in these experiments, and therefore this anion significantly increased in the presence of the electrokineticcould be a significant contributor to the inhibition of bacterial treatment. It has been demonstrated previously that themetabolism. The pH change from neutral to pH 4-5 was activity of sulfur-oxidizing bacteria is stimulated in soil slurriesaccompanied by an alteration in the populations of indig- in the presence of an electric current (21). The soil used inenous sulfur-oxidizing bacteria in the soil. Neutrophiles were these experiments was identical, and the microbial popula-replaced by acidophiles. This pH change also affected the tions would be expected to be similar. The additional effects,solubility of contaminating metal ions, with those such as which may be seen in soil treated with electrokinetics, includecopper and zinc becoming more soluble. The emergence of an increase in microbial activity due to a temperature risethe acidophilic bacteria was therefore combined with the of between 3 and 13 °C. There is also the generation of oxygenmobilization of inhibitory metal ions and the increased at the anode and the subsequent movement of oxygenatedtoxicity of anions. The acidification process therefore slowed water into the soil. Since sulfur oxidation is very oxygen-markedly. dependent, any increase in oxygen in the soil would be Electrokinetic treatment of the soil generated protons at expected to positively affect this process. The stimulation ofthe anode and hydroxyl ions at the cathode. While protons sulfur-oxidizing activity also accompanied an increase inentered the soil and migrated toward the cathode by acetic acid (measured as acetate) movement from theelectromigration, hydroxyl ions were titrated at the cathode electrode and hence its concentration in the soil. A con-with acetic acid to maintain the pH at 4.5 in this compartment. centration of 10 000 mg/kg corresponded to approximatelyThis process led to the soil being rapidly acidified to 670 mM acetate in the pore fluid of the soil. Experiments byapproximately pH 2 within a period of 9 days. At the same Alexander and co-workers (25) have demonstrated that, attime, mobilized copper ions migrated toward the cathode a concentration of 10 mM (pH 3), acetic acid accumulatesby electromigration and entered the cathode chamber where in the cytoplasm of T. ferrooxidans and partially inhibits thethey were pumped through an ion exchange column. oxidation of ferrous iron. The findings of the present studyRemoval of copper was 85% complete within 15 days when in which the soil contained much higher concentrations ofsoil had not been preacidified due to the activity of sulfur- acetic acid clearly demonstrate that these were not inhibitoryoxidizing bacteria. For preacidification experiments, a time to sulfur oxidation by sulfur-oxidizing bacteria. An additionalscale of 90 days was chosen for incubation with sulfur, to experiment, in which a higher current density was usedmaximize sulfate production and acidification based upon resulted in removal of sulfate from the soil to below 1000the results of earlier experiments. The observation that, in mg/kg, is of significance. One of the major drawbacks ofstatic experiments, bacterial activity had ceased after 21 days bioleaching technology is the high production of sulfate fromin the presence of seven contaminating metals, whereas in sulfur/sulfide leading to soil contaminated with sulfate. Theuncontaminated soil, activity continued up to 42 days (and selective and efficient removal of sulfate from soil underpossibly beyond) meant that in studies with a single electrokinetic processing following metal removal maycontaminating metal, a time scale of 90 days might be therefore lead to a cleaner and more effective technology.expected to ensure maximal sulfate generation and acidi- In summary, the combination of preacidification by sulfur-fication. When the soil was allowed to preacidify, there were oxidizing bacteria followed by electrokinetics is cost-effectivetwo marked differences in the overall run parameters. First, in reducing the power input for electrokinetics. The elec-copper removal was achieved with much lower power trokinetic treatment also appears to stimulate the activity ofconsumption. The pH of the soil had already been reduced sulfur-oxidizing bacteria by removal of inhibitory ions andby 2 units due to sulfur oxidation, and therefore the amount other positive effects of the electric current upon soilof hydrogen ions required to acidify the soil to pH 2 and the microbial activity. These synergistic effects are promisingpower required to generate these ions were reduced. In for future experiments in which complex mixtures of metalcommercial application in a field situation, the cost of power ions may be present, and bacteria may be required to mobilizeis a major component of the overall cost of the process. In metals from sulfides. The methodology has potential for aaddition, any reduction in the time scale for electrokinetic range of contaminated sites including former gasworks andremediation will also affect the cost in terms of manpower wastes from mining.and requirements for equipment. By amending soil at anearlier stage with little further intervention, the soil can beprepared for electrokinetic treatment. Acknowledgments A second benefit of the preacidification experiment is This work was supported by the U.K. Department of Tradethat sulfate production by sulfur-oxidizing bacteria was and Industry and the Engineering and Physical Sciencessignificantly stimulated when the electric current was applied. Research Council under the LINK Biological Treatment ofOne of the effects of the electrokinetics was to enable Soils and Water Initiative.